The present invention is directed generally to methods for obtaining paclitaxel and other important taxanes from novel plant sources of taxanes. The present invention is also directed to methods for obtaining paclitaxel and other taxanes by culturing fungal endophytes of these novel sources.
Paclitaxel is the active ingredient in the anticancer drug TAXOL(copyright) (xe2x80x9ctaxolxe2x80x9d) marketed by Bristol Myers Squibb. Taxol has been approved for treatment of ovarian and breast cancers, Kaposi""s sarcoma, and non-small-cell lung cancer. It is also in clinical trial for treatment of several other cancers in combination with other chemotherapeutic agents. Several other uses for paclitaxel have been identified, including possible treatments for psoriasis, polycystic kidney disease, multiple sclerosis, and Alzheimer""s disease. With the increasing number of uses, the need for new sources of paclitaxel is becoming ever more apparent.
Paclitaxel is a complex diterpenoid compound originally extracted from the bark of the Pacific yew tree, Taxus brevifolia. A number of related compounds, collectively known as xe2x80x9ctaxanes,xe2x80x9dare also found in the yew extract. Paclitaxel is currently obtained from various species of yew or is made by partial synthesis from other taxanes also obtained from yew. Taxanes other than paclitaxel are therefore of interest for medicinal use. The chemical structures of six taxanes are shown in FIG. 1.
Yields of taxanes from yew are generally quite small (on the order of milligrams per kilogram of dried plant material). At the present time, there may be enough paclitaxel for FDA-approved uses in chemotherapy. As the numerous clinical trials involving this compound draw to a close, however, it is clear that demand for paclitaxel will increase. Accordingly, it is important to identify alternative sources for this compound.
Laboratory (chemical) synthesis of paclitaxel has been reported from groups led by Holton, Nicoloau, Wender, and Danis hefsky and in patent references directed to chemical synthesis of paclitaxel and other taxanes, or to taxane precursors of paclitaxel:
U.S. Pat. No. 4,468,458 to Sato et al. entitled Two-Functional-Group Terpenoids, Preparation, and Anti-Ulcer Agents.
U.S. Pat. No. 4,814,470 to Colin et al. entitled Taxol Derivatives, Their Preparation, and Pharmaceutical Compositions.
U.S. Pat. No. 4,857,653 to Colin et al. entitled Process for the Preparation of Taxol and 10-Deacetyltaxol.
U.S. Pat. No. 4,924,011 to Denis et al. entitled Process for Preparing Taxol.
U.S. Pat. No. 4,960,790 to Stella et al. entitled Derivatives of Taxol, Pharmaceutical Compositions, and Preparation.
U.S. Pat. No. 5,202,448 to Carver et al. entitled Processes of Converting Taxanes Into Baccatin III.
U.S. Pat. No. 5,232,684 to Blumberg et al. entitled Labeled Resiniferatoxin, Compositions Thereof, and Methods for Using.
U.S. Pat. No. 5,243,045 to Holton et al. entitled Certain Alkoxy Substituted Taxanes and Pharmaceutical Compositions.
Another reference, U.S. Pat. No. 6,043,072 to Croteau et al. entitled Nucleic Acids Encoding Taxus Geranylgeranyl Diphosphate Synthase, claims a nucleic acid sequence and genetic technology for creating nucleic acid sequences that code for taxanes. Many of these syntheses, however, are too complex and costly for commercial use. Accordingly, these syntheses are not a practical method for supplying enough of the drug for all patients. Other references claim cancer treatment methods using taxanes:
U.S. Pat. No. 4,206,221 to Miller et al. entitled Cephalomannine and Its Use in Treating Leukemic Tumors.
U.S. Pat. No. 4,942,184 to Haugwitz et al. entitled Water-Soluble, Antineoplastic Derivatives of Taxol.
U.S. Pat. No. 5,958,741 to Stierle et al. entitled Taxol Production by a Microbe.
Twelve references claim methods for recovering taxanes from plant tissue or from microorganisms obtained from plant tissue:
U.S. Pat. No. 5,019,504 to Christen et al. entitled Production of Taxol or Taxol-like Compounds in Cell Culture.
U.S. Pat. No. 5,322,779 to Strobel et al. entitled Taxol Production by Taxomyces Andreanae.
U.S. Pat. No. 5,445,809 to Strobel et al. entitled Production of Taxol From the Yew Tree.
U.S. Pat. No. 5,451,392 to Strobel et al. entitled Production of Taxol.
U.S. Pat. No. 5,670,663 to Durzan et al. entitled Recovery of Taxanes From Conifers.
U.S. Pat. No. 5,861,302 to Stierle et al. entitled Taxol Production by a Microbe.
U.S. Pat. No. 5,908,759 to Stierle et al. entitled Taxol Production by a Microbe.
U.S. Pat. No. 5,916,783 to Stierle et al. entitled Taxol Production by a Microbe.
U.S. Pat. No. 5,981,777 to Durzan et al. entitled Recovery of Taxanes From Plant Material.
U.S. Pat. No. 6,013,493 to Stierle et al. entitled Taxol Production by a Microbe.
U.S. Pat. No. 6,030,818 to Pagxc3xa9 et al. entitled Bacterial Mass-Production of Taxanes.
U.S. Pat. No. 6,066,748 to Han et al. entitled Process of Extracting TAXOL(copyright) From Taxus Cuspidata.
Researchers such as Strobel, Stierle, Han, and Pagxc3xa9 have shown that several of the bacteria and fungi native to yew trees are capable of making paclitaxel and suggest that fungal cultures may be a viable alternative source for paclitaxel. A number of other bioreactive compounds have also been obtained from plant endophytes isolated from yew and related species.
The following references describe generally the work done by Drs. Strobel and Stierle and their colleagues, which was limited to plants (yew trees) within the genus Taxus:
Stierle, A., G. Strobel, D. Stierle (1993) Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific Yew. Science 260:214-216.
Strobel, G., X. Yang, J. Sears, R. Kramer, R. S. Sidhu, W. M. Hess (1996) Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiology 142:435-440.
Stierle, A., D. Stierle, G. Strobel, G. Bignami, P. Grothaus (1995) Bioactive metabolites of the endophytic fungi of pacific yew, Taxus brevifolia: paclitaxel, taxanes, and other bioactive compounds. In: Taxane Anticancer Agents: Basic Science and Current Status. G. I. Georg, T. T. Chen, I. Ojima, D. M. Vyas, eds. ACS Symposium Series 583, Wash. D.C., pp. 81-97.
U.S. Pat. No. 5,451,392 to Strobel et al. entitled Production of Taxol.
U.S. Pat. No. 5,445,809 to Strobel et al. entitled Production of Taxol From the Yew Tree.
U.S. Pat. No. 5,322,779 to Strobel et al. entitled Taxol Production by Taxomyces Adreanae. 
U.S. Pat. No. 6,013,493 to Stierle et al. entitled Taxol Production by a Microbe.
U.S. Pat. No. 5,958,741 to Stierle et al. entitled Taxol Production by a Microbe.
U.S. Pat. No. 5,916,783 to Stierle et al. entitled Taxol Production by a Microbe.
U.S. Pat. No. 5,908,759 to Stierle et al. entitled Taxol Production by a Microbe.
U.S. Pat. No. 5,861,302 to Stierle et al. entitled Taxol Production by a Microbe.
Other researchers, such as Don J. Durzan and Frank Ventimiglia, discovered taxane-producing plants outside of the genus Taxus but still within the botanical order containing the genus Taxus. U.S. Pat. No. 5,670,663 to Durzan et al. (the xe2x80x9cDurzan referencexe2x80x9d) is directed to recovery of taxanes from conifers. The Durzan reference is significant for broadening the state of the art with regard to plant species from which taxanes may be obtained. Before the work of Durzan et al., it was thought that taxanes could be obtained only from plants of the genus Taxus. The Durzan reference suggested that taxane-producing plants are limited to gymnosperms of the order of conifers, including the genus Taxus. Neither the work of Strobel and Stierle et al. nor the work of Durzan et al. suggests that taxanes could ever be discovered in angiosperms or in other gymnosperms.
Background information on the inventor""s work may be found in the following publication coauthored by the inventor:
Hoffman, A., W. Khan, J. Worapong, G. Strobel, D. Griffen, B. Arbogast, D. Barofsky, R. B. Boone, L. Ning, P. Zheng, L. Daley (1998) Bioprospecting for Taxol in angiosperm plant extracts: Using high performance liquid chromatography-thermospray mass spectrometry to detect the anticancer agent and its related metabolites in filbert trees. Spectroscopy 13:22-32.
Finally, the following references generally describe various aspects of the art:
Wani, M. C., W. H. Taylor, M. E. Wall, P. Coggon, A. T McPhail (1971) Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc. 93:2325-2327.
Difco Laboratories (1953) Difco Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Procedures, 9th ed., pp. 64-65 and 242-246. Difco Laboratories, Detroit, Mich.
Each of the above scientific and patent references is incorporated into this application in its entirety by reference.
Taxanes have been isolated from various conifers, by Strobel and Stierle, among others. According to the method of Strobel and Stierle for isolating taxanes, plant material from the taxane-producing conifer is ground, and the taxane is isolated from this ground plant material. This method has the disadvantages of low yield and the introduction of contaminants from the surface of the plant material. A method for isolating a taxane from plant material that overcomes these disadvantages is needed.
As discussed above, taxanes were first discovered in yew trees of the genus Taxus. Later, other researchers determined that taxanes are also produced in other conifers and in particular strains of hazelnut trees of the genus Corylus, a member of the order of angiosperms, plants that produce seeds. See Hoffman et al., above. To date, hazelnut is the only angiosperms in which taxanes have ever been found to be produced.
Strobel and Stierle further disclosed that taxanes may be isolated from a certain endophytic fungus, Taxomyces andreanae, that lives within yew trees. Strobel and Stierle later disclosed that several other endophytic fungi of yew trees produce taxanes. Likewise, Hoffman et al. disclose that endophytic fungi from the hazelnut strains that produce taxanes themselves produce taxanes when grown in culture.
Because taxanes are produced in very low concentrations in these sources, a significant need exists to find other sources of taxanes if possible, especially of paclitaxel.
In this specification, the term xe2x80x9ctaxanexe2x80x9drefers generally to any or all of the taxanes, a known class of chemical compounds having antitumor properties, whether now known or discovered in the future. The use of any specific taxane, such as xe2x80x9cpaclitaxel,xe2x80x9d refers solely to that particular taxane.
The present invention is directed generally to methods for obtaining paclitaxel and other important taxanes from novel plant sources. The present invention is also directed to methods for obtaining paclitaxel and other taxanes by culturing fungal endophytes of these novel sources.
It has been discovered that the yield of taxanes isolated from plant material from a plant that produces taxanes may be increased by sterilizing the surface of the plant material before extracting the taxane. It has been further discovered that the yield of taxanes that is isolated from such plant material may be increased by not grinding the plant material before extracting. Preferably, the plant material is surface-sterilized and is not ground before the extraction of taxanes.
One embodiment of the invention is a method for extracting a taxane from a part of a taxane-producing plant. The method of the invention includes sterilizing the surface of the plant part before extracting the taxane.
In another embodiment, the invention is a method for extracting a taxane from a part of a taxane-producing plant. The method of the invention omits grinding the plant part before extracting the taxane.
Preferably, the method of the invention includes sterilizing the surface of the plant part and omits grinding the plant part before extracting the taxane.
It has been discovered in accordance with the invention that taxanes are produced and may be isolated from angiosperms other than hazelnut trees of the genus Corylus. Surprisingly, the inventor has discovered that taxanes are produced in a wide variety of angiosperms. This exciting discovery opens the possibility for the first time that taxanes will be abundantly found in many species and that future supplies of taxanes will be available to meet expected future demand.
In another embodiment, the invention is a method for extracting a taxane from a plant part of an angiosperm other than a hazelnut tree. Preferably, the taxane is extracted from a part of the nonhazelnut angiosperm following surface sterilization of the part with or without grinding the part before the extraction of the taxane.
It has been further discovered that taxanes are produced by endophytic fungi of angiosperms other than those of hazelnut trees of the genus Corylus. This discovery is likewise an exciting one that opens the possibility that taxanes will be abundantly available to meet future needs.
In another embodiment, the invention is a method for obtaining taxane by culturing a taxane-producing fungus that is an endophyte of an angiosperm other than hazelnut of the genus Corylus and obtaining the taxane produced by the fungus. Preferably, the endophytic fungus is a fungus that is not a member of a taxonomic species that is an endophyte of a gymnosperm or of hazelnut. Most preferably, the endophytic fungus according to the method of the invention is of the genus Alternaria, a genus of fungus that has not until now been discovered to produce a taxane.
In another embodiment, the invention is a method for isolating a taxane-producing endophytic fungus from a plant. In accordance with this embodiment, a plant part is surface-sterilized before placing the plant part in contact with a growth medium capable of supporting fungal growth. Preferably, the plant part is not ground before contacting the plant part with the growth medium.
In another embodiment, the invention is a novel fungus that has been discovered to produce taxane.
The foregoing and other objectives, features, and advantages of the invention will be more readily understood upon consideration of the following detailed description of the invention, taken in conjunction with the accompanying drawings.